identification of active compounds and their metabolites by high-performance liquid...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2009; 23: 2724–2732

) DOI: 10.1002/rcm.4179
Published online in Wiley InterScience (www.interscience.wiley.com
Identification of active compounds and their metabolites

by high-performance liquid chromatography/electrospray

ionization Fourier transform ion cyclotron resonance

mass spectrometry from Xiao-xu-ming decoction (XXMD)

Yilin Wang1, Chunguang Ding1, Kehe Du1,2, Yao Xiao1, Caisheng Wu1, Jinlan Zhang1,3*,

Hailin Qin1 and Guanhua Du1

1Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, P.R. China2Liaoning Shihua University, Liaoning 11300, P.R. China3Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Institute of Materia Medica, Chinese Academy

of Medical Sciences & Peking Union Medical College, Beijing 100050, P.R. China

Received 13 April 2009; Revised 25 June 2009; Accepted 27 June 2009

*CorrespoChinese ACollege, BE-mail: zContract/contract/

Xiao-xu-ming decoction (XXMD) prescription is a traditional Chinese prescription that has been

widely used to treat theoplegia and the sequela of theoplegia. Modern pharmacological research

has also indicated that the active fraction from XXMD is able to treat cardiovascular diseases

and Alzheimer’s disease. In the study reported here, high-performance liquid chromatography

coupled with Fourier transform ion cyclotron resonance mass spectrometry (HPLC/FTICR-MS)

was developed to identify active compounds and their metabolites after oral administration of

active fraction from Xiao-xu-ming decoction to rats, using parent mass list triggered data-dependent

multiple-stage mass analysis at a resolving power of 100 000 in the external calibration mode. The

mass accuracies obtained for full-scan MS were within 2ppm in most cases. Fifteen constituents

were identified in the active fraction from XXMD and the biological samples of rats. The fragmenta-

tion behaviors of these constituents were summarized which would be helpful for structural

characterization. The profiles of the constituents in the active fraction and biological samples

of rats were obtained which provided us with much information for a better understanding

of the chemical basis of the pharmacologic actions of XXMD. Copyright # 2009 John Wiley & Sons,

Ltd.

Xiao Xu Ming-Decoction (XXMD) prescription is a tradi-

tional Chinese prescription that was firstly recorded in

‘QianJinYaoFang’ which was written by Chinese ancient

Si-Miao Sun of the Tang Dynasty. The formula consists of

twelve crude drugs including Saposhnikovia divaricata

(Turcz.) Schischk., Scutellaria baicalensis Georgi, Paeonia

lactiflora Pall., Glycyrrhiza uralensis Fisch., Zingiber officinale

Rosc., Stephania tetrandra S. Moore, Panax ginseng C. A. Mey.,

Cinnamomum cassia Presl, Prunus armeniaca L. var. ansu

Maxim., Ephedra sinica Staph, Ligusticum chuanxiong Hort.,

Aconitum carmichaeli Debx. in a ratio of 3:3:3:3:3:3:6:6:6:9:9:9

on a dry weight basis. The prescription has attracted a great

deal of attention for its pharmaceutical and therapeutic uses.

The active fraction was screened byWang et al.1 and showed

similar pharmacological effects to XXMD. It is effective in

ndence to: J. L. Zhang, Institute of Materia Medica,cademy of Medical Sciences & Peking Union Medicaleijing 100050, P.R. China.

[email protected] sponsor: National Nature Science Foundation;grant number: 30630073.

treating theoplegia and the sequela of theoplegia. Modern

pharmacological work has also indicated that the active

fraction is able to treat cardiovascular diseases and

Alzheimer’s disease.2,3

Fourier transform ion cyclotron resonance mass spec-

trometry (FTICR-MS) is a mass spectrometry technique

which has been developed rapidly in the last 30 years. In

1974, Comisarow and Marshall4,5 adapted Fourier transform

methods to ICR spectrometry and built the first FTMS

instrument. Since that time, FTMS mass spectrometers with

the highest mass accuracy and mass resolving power6 have

received increasingly attention and become efficient tools for

various analyses including proteomics analyses,7 accurate

mass measurements for drug discovery,8 drug mixture

analyses,9 analyses of trace impurities in a drug substance,10

toxicology and forensic sciences,11 microbiology,12 and most

recently, metabonomics.13–17

As the active fraction of XXMD is prepared from twelve

medicinal herbs and each of them contains very complex

chemical components, it is very important to identify and

characterize the constituents present in the active fraction

Copyright # 2009 John Wiley & Sons, Ltd.

Active compounds and their metabolites in XXMD 2725

and the metabolites in vivo for a better understanding of

its mechanism of action. In the work reported herein, a

high-performance liquid chromatography (HPLC) separ-

ation prior to LTQ/FTICR-MS was employed to profile

parent constituents in the active fraction from XXMD, as well

as the metabolites after oral administration of the active

fraction to rats. The accuracy of HPLC/FTICR-MS m/z value

measurements were within 2ppm in most cases. Besides,

multistage MS/MS analysis (MSn) was applied to provide

further information about the structures of the compounds

under analysis. So a novel method for the rapid identification

and characterization of constituents in complex extract and

their metabolites was developed using the combination of

high mass accuracy and fragmentation behavior. As a result,

15 constituents were totally identified in the active fraction

from XXMD and biological samples from rats.

EXPERIMENTAL

Chemicals, reagents, and materialsPaeoniflorin (1), prim-o-glucosylcimifugin (2), cimifugin (3),

40-O-b-D-glucosyl-5-O-methylvisamminol (4), baicalin (5),

baicalein (10), glycyrrhizic acid (11), glycyrrhetinic acid (12),

wogonin (13), and chrysin (14) were purchased from the

National Institute for Control of Pharmaceutical and

Biological Products (Beijing,China). Liquiritigenin (6) was

obtained from the Dalian Fusheng Pharmaceutical Co., Ltd.

Wogonoside (9) was ordered from the Shanghai Usea Biotech

Co., Ltd. Oroxylin A-7-O-glucuronide (8) and oroxylin A (15)

were gifts from Professor Hailin Qin. 5-O-Methylvisammiol

(7) was isolated and purified in our laboratory. The purity of

all compounds was greater than 99% (by HPLC). The

structures of these materials were confirmed by UV, MS,1H NMR, and 13C NMR analyses. The structures are shown

in Scheme 1.

Acetonitrile (LC/MS reagent grade) was supplied by

Mallinckrodt Baker, Inc. (Phillipsburg, NJ, USA). Deionized

water was purified using a water purification system

(Millipore, Billerica, MA, USA). Analytical grade ethyl

acetate, methanol and acetic acid were purchased from

Beijing Chemical Corp. (Beijing, China). The active fraction of

XXMD prescription was also a kind gift from Professor

Hailin Qin.

Preparation of the dosed solution ofthe active fractionThe orally administrated solution of the active fraction

powder was dissolved in deionized water at the concen-

tration of 250mg/mL, adding 0.5% Tween 80 as solution

adjuvant.

ChromatographyA Thermo Scientific Surveyor LC Plus system equipped with

a Surveyor MS pump plus and a Surveyor autosampler was

used to carry out the assay. The samples were separated on a

HYPERSIL C18 column (100� 2.1mm, 5mm) protected by a

ZORBAX SB-C18 guard column (12.5� 2.1mm, 3.5mm). The

mobile phase consisted of acetonitrile (A) and 0.4% (v/v)

acetic acid (B) delivered at a flow rate of 0.8mL/min with the


following gradient program: starting with 15% A, then

reaching 25%A at 20min, reaching 30%A at 35min, reaching

40% A at 40min and maintaining 40% A until 50min, then

reaching 60% A at 60min and maintaining 60% A until

70min. The systemwas then returned to the initial conditions

within 30 s, and the column was reconditioned for 9.5min.

The column temperature was maintained at 308C and the

sample injection volume was 10mL.

Mass spectrometryA Thermo Scientific LTQ FT mass spectrometer was

connected to the Thermo Scientific Surveyor LC Plus system

via an electrospray ionization (ESI) interface. The column

effluent was split in a ratio of 3:1, so that 200mL/min entered

the source of the mass spectrometer. The operating

parameters in the positive ion mode were as follows:

collision gas, ultrahigh-purity helium (He); nebulizing gas,

high-purity nitrogen (N2); ion spray voltage, 3.5 kV; capillary

temperature, 3008C; capillary voltage, 40V; sheath gas flow

rate, 35 (arbitrary units); auxiliary gas flow rate, 10 (arbitrary

units); sweep gas flow rate, 5 (arbitrary units); and tube lens,

120V. Mass spectra were recorded in a mass range ofm/z 100

to 1500 at a resolving power of 100 000 with data-dependent

MS/MS analysis triggered by the most abundant ions from

the parent mass list of predicted compounds (mass list of the

positive ion mode: 823, 503, 471, 469, 461, 453, 447, 307, 291,

285, 271, 257, 255), followed by MS/MS/MS analysis on the

most abundant product ions. Collision-induced dissociation

(CID) was conducted with an isolation width of 1 Da.

Xcalibur software (version 2.0; Thermo Scientific) was

employed for data acquisition and reduction after HPLC/

FTICR-MS analysis.

AnimalsMale Wistar rats (190� 20 g) were obtained from the

Laboratory Animal Center of the Chinese Academy of

Medical Sciences & Peking Union Medical College (Beijing,

China). The animals were kept in a fully acclimatized room

for 3 days before starting the experiment and had free access

to routine diet and water. Prior to the experiment rats were

fasted in ametabolic cage and given only physiological saline

for 24 h. Then they were orally given the active fraction from

Xiao-xu-ming decoction at a dose of 400mg/200 g body

weight. All protocols and procedures involving animals

were approved by the Animal Care and Welfare Committee

of Institute of Materia Medica, Chinese Academy of Medical

Sciences & Peking Union Medical College (Beijing, China).

Urine and feces were collected after administration during

different periods thereafter (0–12, 12–24, and 24–48 h). The

amounts of urine and feces during each period were

recorded, and samples were stored at �208C until assay.

Blood samples were obtained from the abdominal aorta

according to the specific schedule (5, 20, 45, 60, 90, 120, 180,

240, 300, 360, 420, 450, 480, 510, 540, 600, 720, 960, 1440min)

after the oral administration of the active fraction to rats.

Plasmawas separated by centrifugation at 765 g for 5min. All

plasma samples were stored at �208C until the assay was

performed.


DOI: 10.1002/rcm

Scheme 1. Chemical structures of the 15 identified constituents in the active fraction from XXMD

and their metabolites in rats.

2726 Y. L. Wang et al.

Sample preparation

Sample preparation from plasma and urineAll plasma or urine samples were centrifuged at 1721 g for

10min and then divided into two portions, respectively. For

one portion, aliquots of 1mL of plasma or urine supernatants

were placed into 5mL conical plastic test tubes and 1mL

amounts of ethyl acetate were added. Next, the test tubes

were vortex-mixed vigorously for 90 s and centrifuged at

1721 g for 10min. The extraction was repeated twice. The

supernatants were combined and evaporated to dryness at

308C under reduced pressure. For another portion, aliquots

of 1mL of urine or plasma supernatants were placed into

10mL conical plastic test tubes and 6mL amounts of

methanol were added. Next, the test tubes were vortex-


mixed vigorously for 90 s and centrifuged at 1721 g for

10min. The supernatants were evaporated to dryness at 308Cunder reduced pressure. All residues were reconstituted in

0.2mL amounts of methanol. Each sample was then filtered

through a 0.45mm nylon filter film. Aliquots of 10mL were

injected into the HPLC/FTICR-MS system.

Sample preparation from fecesAll feces samples were weighed and completely ground

with ten times their volume of physiological saline and

the mixtures centrifuged at 1721 g for 10min. Then the

supernatants were divided into two portions which

were treated with ethyl acetate and methanol, respectively.

The subsequent procedures were as described above for

plasma and urine.


DOI: 10.1002/rcm


RESULTS AND DISCUSSION

Structural characterization of constituents in theactive fraction from XXMD

The MS and MSn experiments using LTQ/FTICR-MS were

first performed to investigate the fragmentation character-

istics of the constituents in the active fraction from XXMD

and the relative standards. These fragmentation patterns and

the corresponding masses of fragment ions are sufficient to

determine the chemical structures of the compounds in the

active fraction, and furthermore to identity the constituents

of the biological specimens from rats.

In this study, a Thermo LTQ FTICR-MS systemwas chosen

to carry out the analysis. The Thermo LTQ FTICR-MS system

combines a linear ion trap (IT) mass spectrometer with an

FTICR analyzer.18 The linear IT, with greater ion storage

capacity than conventional three-dimensional (3D) IT

devices, is a fully operational MS detector, which can store,

isolate, and fragment ions. And all the ion handling, selection

and excitation capabilities of the IT can be used to prepare

ions for analysis in the ICR cell. The combination can

enhance ion accumulation and improve the duty cycle,

therefore making it well suited for LC analysis and

meanwhile providing excellent performance and capabilities

to the FT mass spectrometers. In this paper, the mass

accuracies of the compounds in the active fraction obtained

for full-scan MS spectra were within 4ppm. In most cases,

the errors were within 1 ppm, as listed in Table 1.

The HPLC/FTICR-MS analysis of the active fraction is

shown in Fig. 1. Constituents in the active fraction were

profiled in one chromatographic run with parent mass list

triggered data-dependent multiple-stage mass analysis. By

comparing with the reference compounds, 14 constituents

including eight flavonoids (peak nos. 5, 6, 8, 9, 10, 13, 14, 15),

Table 1. Mass data from the fifteen compounds detected in the a

No.Rt

(min) [MþH]þProposedformula

Error(ppm) Identified compoun

1 8.28 503.15182� C23H28O11Naþ �1.12 paeoniflorin2 8.76 469.17093 C22H29O

þ11 1.05 prim-o-glucosylcimi

3 13.45 307.11761 C16H19Oþ6 �0.02 cimifugin

4 15.95 453.17578 C22H29Oþ10 0.57 40-O-b-D-glucosyl-5

methylvisamminol5 20.96 447.09256 C21H19O

þ11 0.83 baicalin

6 24.55 257.08084 C15H13Oþ4 �0.02 liquiritigenin

7 24.7 291.12286 C16H19Oþ5 0.55 5-O-methylvisammi

8 26.60 461.10706 C22H21Oþ11 �1.69 oroxylin A 7-O-gluc

9 29.48 461.10776 C22H21Oþ11 �0.17 wogonoside

10 40.97 271.05951 C15H11Oþ5 �2.18 baicalein

11 46.06 823.41156 C42H63Oþ16 0.60 glycyrrhizic acid

12 48.14 471.34833 C30H47Oþ4 3.06 glycyrrhetinic acid

13 48.64 285.07523 C16H13Oþ5 �1.82 wogonin

14 49.92 255.06522 C15H11Oþ4 0.14 chrysin

15 51.49 285.07565 C16H13Oþ5 �0.35 oroxylin A

�The ions at m/z 503.15182 and 503.15238 were [MþNa]þ.a XX, PL, UR and FE represent XXMD, plasma, urine and feces, respectivND: not detected, þ: detected. Rt: retention time.


three chromones (peak nos. 2, 3, 4), two triterpenes (peak nos.

11, 12) and one monoterpene (peak no. 1) were identified, as

listed in Tables 1 and 2, respectively.

HPLC/LTQ-MSn investigation of compounds 5, 6,8, 9, 10, 13, 14, 15These eight compounds are all flavonoids. The common

features of ESI-MS/MS data of [MþH]þ ions observed in this

work were the loss of CO (28 Da) and H2O (18 Da). The

compounds containing methoxyl groups could also give an

ion loss of CH3 (15 Da). Also, the retro-Diels-Alder (RDA)

fragmentation reaction could be observed. In previous

studies,19–23 flavonoids were mostly investigated by MS in

the negative mode. However, the common features of these

reported data are similar to that obtained in the positive

mode in our work.

Baicalin (5) displayed an [MþH]þ ion atm/z 447.09256 and

gave an ion at m/z 271 in the MS2 spectrum as the base peak

from the neutral loss of a glucuronic acid. This neutral loss

can be used for the identification of O-glucuronides.

Baicalein (10) gave an [MþH]þ ion at m/z 271.05951. The

MS2 spectrum of baicalein yielded product ions at m/z 253

(base peak) and 225, which were from the loss of H2O and

CO. Besides, the ion atm/z 169 and 103 was produced from a

RDA fragmentation reaction cleaved at the C-ring. Lliquir-

itigenin (6) exhibited an [MþH]þ ion at m/z 257.08084. In the

MS2 spectrum, the product ions at m/z 239 and 137 were

formed by the loss of H2O and the RDA fragmentation

reaction, respectively. The fragment ion at m/z 211 in the

MS3 spectrumwas attributed to the loss of CO from the ion at

m/z 239, whichwas themost abundant product ion in theMS2

spectrum. Oroxylin A 7-O-glucuronide (8) and wogonoside

(9) are a pair of isomers, both having glucuronic acid

groups on the A-ring and showing an [MþH]þ ion atm/z 461

ctive fraction from XXMD, plasma, urine and feces

ds Plant source

Distributiona

XX PL UR FE

Paeonia lactiflora Pall. þ þ þ NDfugin Saposhnikovia divaricata (Turcz.)

Schischk.þ þ þ ND

Saposhnikovia divaricata (Turcz.)Schischk.

þ þ þ þ

-O- Saposhnikovia divaricata (Turcz.)Schischk.

þ ND ND ND

Scutellaria baicalensis Georgi þ þ ND ND

Glycyrrhiza uralensis Fisch. þ þ þ þol Saposhnikovia divaricata (Turcz.)

Schischk.ND þ þ þ

uronide Scutellaria baicalensis Georgi þ þ þ ND

Scutellaria baicalensis Georgi þ þ þ ND

Scutellaria baicalensis Georgi þ ND þ þGlycyrrhiza uralensis Fisch. þ þ þ þGlycyrrhiza uralensis Fisch. þ þ þ þScutellaria baicalensis Georgi þ þ þ þScutellaria baicalensis Georgi þ ND þ þScutellaria baicalensis Georgi þ þ þ þ

ely.


DOI: 10.1002/rcm

Figure 1. The extracted ion chromatograms of the active fraction fromXXMD. The numbers assigned to compounds are the same

as in Scheme 1.

Copyright # 2009 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2009; 23: 2724–2732

DOI: 10.1002/rcm


Table 2. HPLC/LTQ-MSn data from the compounds detected in the active fraction from XXMD, plasma, urine and feces

No. Name HPLC/LTQ-MSn Ref.

1 paeoniflorin MS2[503]: 381, 341, 307, 219, 185 26,27MS3[381]: 363

2 prim-o-glucosylcimifugin MS2[469]: 307, 289, 259, 235 —3 cimifugin MS2[307]: 289, 259, 235 24

MS3[235]: 217, 2074 40-O-b-D-glucosyl-5-O-methylvisamminol MS2[453]: 291, 273 —

MS3[291]: 273, 2435 baicalin MS2[447]: 271 19,216 liquiritigenin MS2[257]: 239, 211, 163, 147, 137 19,22

MS3[239]: 211, 183, 165MS3[147]: 119

8 oroxylin A 7-O-glucuronide MS2[461]: 285, 270 —MS3[285]: 270

9 wogonoside MS2[461]: 285, 270 20,21MS3[285]: 270

10 baicalein MS2[271]: 253, 225, 169, 103 19,2011 glycyrrhizic acid MS2[823]: 471 2512 glycyrrhetinic acid MS2[471]: 453, 435, 389 —13 wogonin MS2[285]: 270, 183, 103 20

MS3[270]: 252, 242, 22414 chrysin MS2[255]: 237, 227, 153, 103 19,2015 oroxylin A MS2[285]: 270, 183, 103 19,23

MS3[270]: 252, 242, 224


(8:m/z 461.10706, 9:m/z 461.10776). The MS2 andMS3 spectra

of the two compounds were highly similar to each other,

except for the relative abundance of ions. The ion at m/z 285

in the MS2 spectrum was generated by the loss of glucuronic

acid from the molecular ion atm/z 461, and the ion atm/z 270

in the MS3 spectrum was formed by the loss of CH3 from the

ion at m/z 285. It was hard to distinguish these two isomers

only by MS spectra, but it was easier to identify them by

comparing their retention behaviors with the help of the

available reference standards. Similarly, wogonin (13) and

oroxylin A (15) are also a pair of isomerswhich gave the same

[MþH]þ ion at m/z 285 and showed an ion at m/z 270 as the

base peak from the loss of CH3. They were finally identified

by comparing the two compounds with the reference

standards. Chrysin (14) displayed an [MþH]þ ion at m/z

255.06522. In theMS2 experiment the [MþH]þ ion underwent

the RDA fragmentation reaction to yield product ions at m/z

153 and 103.

HPLC/LTQ-MSn investigation of compounds 2,3, 4prim-o-Glucosylcimifugin (2), cimifugin (3) and 40-O-b-D-

glucosyl-5-O-methylvisamminol (4) are all chromones. prim-

o-Glucosylcimifugin (2) gave an [MþH]þ ion at m/z

469.17093. In the MS2 spectrum, the [MþH]þ ion lost a

glucose group and transformed into cimifugin (3), giving a

product ion at m/z 307. Cimifugin (3) displayed an [MþH]þ

ion atm/z 307.11761. In theMS2 spectrum, the product ions at

m/z 289, 259 and 235 (base peak) were formed by the loss

of H2O, HCHO and C3H4O2 (72 Da) from the [MþH]þ ion at

m/z 307, respectively. The ion at m/z 235 then lost H2O and

CO to produce the ions at m/z 217 and 207 in the MS3

spectrum. 40-O-b-D-Glucosyl-5-O-methylvisamminol (4) dis-

played an [MþH]þ ion at m/z 453.17578. Its fragmentation


patterns were similar to that of cimifugin (3) and displayed

the characteristic product ions at m/z 291 and 273 in the MS2

spectrum.

HPLC/LTQ-MSn investigation of compounds11, 12Glycyrrhizic acid (11) and glycyrrhetinic acid (12) are both

triterpenes. Glycyrrhizic acid (11) showed an [MþH]þ ion at

m/z 823.41156 and can successively lose two glucuronic acid

groups and then transform into glycyrrhetinic acid (12).

Glycyrrhetinic acid (12) afforded an [MþH]þ ion at m/z

471.34833. In the MS2 spectrum of glycyrrhetinic acid (12),

the ions at m/z 453 and 435 were formed by the successive

loss of H2O and 2 H2O.

HPLC/LTQ-MSn investigation of compound 1Paeoniflorin (1) is a monoterpene compound which gave an

[MþNa]þ ion at m/z 503.15265. The ions at m/z 381 (base

peak), 341 and 307 in the MS2 spectrum were produced from

the losses of a benzoic acid, a glucose and a parent nucleus,

respectively. The fragment ion at m/z 363 was then produced

from the loss of H2O from the ion at m/z 381 in the MS3

spectrum.

Identification of constituents in the plasma ofratsThe MS and MSn data of the plasma which were obtained

from the rats treated with the active fraction from XXMD

(Tables 1 and 2) were firstly compared to the blank plasma

samples to ensure that the constituents detected did not exist

in the blank samples.

Then the data of the plasma were compared with that of

the active fraction and the standards, and a total of eleven

constituents were detected in the plasma of rats, as can be


DOI: 10.1002/rcm

Figure 2. The extracted ion chromatograms of the plasma of rats treated with ethyl acetate. The

numbers assigned to compounds are the same as in Scheme 1.


seen from Figs. 2 and 3. The mass accuracies of the

constituents in the plasma obtained for full-scan MS spectra

were within 1ppm in most cases. Glycyrrhetinic acid (12),

however, showed an error of 5.59 ppm as a special case. The

main differences between the compounds found in the

plasma and the active fractionwere that 40-O-b-D-glucosyl-5-

O-methylvisamminol (4), baicalin (5), baicalein (10) and

chrysin (14) were not detected in the plasma. However, 5-O-

methylvisammiol (7), which was formed by the loss of a

glucose from 40-O-b-D-glucosyl-5-O-methylvisamminol (4)

and gave an [MþH]þ ion at m/z 291.12286, caught our

attention. We also found that the compounds detected both

in plasma and the active fraction had different relative

amounts, which indicated that these compounds had

different abilities to be absorbed into the plasma attributed

to their different physicochemical characteristics.

Identification of constituents in the urine ofratsThe strategy used for the study of urine of rats was the same

as that of plasma. By comparing the MS and MSn data of the

urine (Tables 1 and 2) with that of the blank urine samples

and the active fraction, a total of 14 constituents were


detected in the urine of rats (Table 1). The mass accuracies of

the constituents detected in the urine obtained for full-scan

MS spectra were within 1 ppm in most cases, except for

cimifugin (3, error �7.54 ppm) and glycyrrhizic acid (11,

error 6.83 ppm). The reason for this high error may be due to

theweak peak intensity of cimifugin (3) and glycyrrhizic acid

(11) in rat urine.

Identification of constituents in the feces of ratsBy comparing the MS and MSn data of the feces (Tables 1

and 2) with that of the blank feces samples and the active

fraction, a total of nine constituents were detected in the feces

of rats (Table 1). The mass accuracies of the constituents

detected in the feces obtained for full-scan MS spectra were

within 3ppm in most cases, except for glycyrrhizic acid (11,

error�12.59 ppm). The reason for this high error may be also

due to the weak peak intensity of glycyrrhizic acid (11) in rat

feces. Compared with the analytical results from the urine, it

was found that some glycosides with higher polarity, such as

cimifugin (3), baicalin (5) and wogonoside (9) were all not

detected in the feces. The result demonstrated that the

compounds that were present in the feces were mainly the

compounds with lower polarity.


DOI: 10.1002/rcm

Figure 3. The extracted ion chromatograms of the plasma of rats treated with methanol. The

numbers assigned to compounds are the same as in Scheme 1.


CONCLUSIONS

In this paper, an HPLC/FTICR-MS method was developed

to profile parent constituents in the active fraction from

XXMD, as well as biological samples after oral adminis-

tration of the active fraction to rats. Accurate mass

measurement at a resolving power of 100 000 indicated that

the technique was capable of providing mass accuracy

within 2ppm in most cases in the external calibration mode.

The results suggest that HPLC/FTICR-MS is a valuable

analytical tool in compound identification because it can

provide robust accurate mass andMS/MS determinations of

the compounds. The valuable information is helpful for the

rapid confirmation of expected metabolites and elucidation

of the structures of unusual or unexpected metabolites. As a

result, 15 constituents were totally identified in the active

fraction from XXMD and the biological samples from rats.

The fragmentation behaviors of these constituents were

summarized which would be helpful for structural charac-

terization. The profiles of the constituents in the active

fraction and the biological samples of rats were obtained

which could provide us with much more information for

better understanding of pharmacologic actions of XXMD

from the chemical viewpoint.


AcknowledgementsWe are grateful for financial support from the National

Nature Science Foundation of China (No. 30630073).

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